A real-time PCR assay for accurate quantification of the individual members of the Altered Schaedler Flora microbiota in gnotobiotic mice.
J Microbiol Methods
; 135: 52-62, 2017 04.
Article
em En
| MEDLINE
| ID: mdl-28189782
ABSTRACT
Changes in the gastrointestinal microbial community are frequently associated with chronic diseases such as Inflammatory Bowel Diseases. However, understanding the relationship of any individual taxon within the community to host physiology is made complex due to the diversity and individuality of the gut microbiota. Defined microbial communities such as the Altered Schaedler Flora (ASF) help alleviate the challenges of a diverse microbiota by allowing one to interrogate the relationship between individual bacterial species and host responses. An important aspect of studying these relationships with defined microbial communities is the ability to measure the population abundance and dynamics of each member. Herein, we describe the development of an improved ASF species-specific and sensitive real-time quantitative polymerase chain reaction (qPCR) for use with SYBR Green chemistry to accurately assess individual ASF member abundance. This approach targets hypervariable regions V1 through V3 of the 16S rRNA gene of each ASF taxon to enhance assay specificity. We demonstrate the reproducibility, sensitivity and application of this new method by quantifying each ASF bacterium in two inbred mouse lines. We also used it to assess changes in ASF member abundance before and after acute antibiotic perturbation of the community as well as in mice fed two different diets. Additionally, we describe a nested PCR assay for the detection of lowly abundant ASF members. Altogether, this improved qPCR method will facilitate gnotobiotic research involving the ASF community by allowing for reproducible quantification of its members under various physiological conditions.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Contexto em Saúde:
3_ND
Problema de saúde:
3_zoonosis
Assunto principal:
Bactérias
/
DNA Bacteriano
/
Reação em Cadeia da Polimerase em Tempo Real
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Microbioma Gastrointestinal
/
Vida Livre de Germes
Tipo de estudo:
Diagnostic_studies
/
Prognostic_studies
Limite:
Animals
Idioma:
En
Revista:
J Microbiol Methods
Ano de publicação:
2017
Tipo de documento:
Article
País de afiliação:
Estados Unidos